Chen J, Stickles R J, Daichendt K A
Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, Canada.
Hum Gene Ther. 1994 Apr;5(4):429-35. doi: 10.1089/hum.1994.5.4-429.
We have developed a novel, highly efficient DNA delivery system to accomplish gene transfer through the asialoglycoprotein receptor-mediated endocytosis pathway. Natural nuclear DNA-binding proteins, the histones (H1, H2a, H2b, H3, and H4), were modified and used as receptor-targeted DNA carriers. Galactosylated with a coupling agent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the histones and albumin were conjugated to DNA and then used to transfect HepG2 cells, which display the asialoglycoprotein receptor. The extent of galactosylation was determined for all histone subgroups and albumin with 14C-labeled galactose. A reporter gene for the bacterial chloramphenicol acetyltransferase (CAT), under the control of the 5' long terminal repeat (LTR) of Rous sarcoma virus, was used for comparisons of transfection efficiency of various carrier proteins. The CAT activity resulting from histone H1-mediated transfection was 1.66 unit per 10(6) cells, the highest among histone subgroups. The galactyosylated histone H1 was also eleven times more effective than the asialo-orosomucoid-polylysine. Ten galactosyl units are attached to histone H1 by the galactosylation reaction. Differences in the extent of galactosylation could not explain different transfection efficiencies among various proteins studied in this report. Treatment with galactose oxidase abolished the transfection ability of both the galactosylated histone H1 and asialo-orosomucoid. The intrinsic DNA-binding domains and nuclear location signal sequences are unique to histones as receptor-targeted DNA carriers, and are advantageous for effective gene delivery.
我们开发了一种新型高效的DNA递送系统，以通过去唾液酸糖蛋白受体介导的内吞途径完成基因转移。天然核DNA结合蛋白，即组蛋白（H1、H2a、H2b、H3和H4），经过修饰后用作受体靶向DNA载体。组蛋白和白蛋白用偶联剂1-乙基-3-（3-二甲基氨基丙基）碳二亚胺进行半乳糖基化，与DNA结合，然后用于转染表达去唾液酸糖蛋白受体的HepG2细胞。用14C标记的半乳糖测定所有组蛋白亚组和白蛋白的半乳糖基化程度。在劳氏肉瘤病毒5'长末端重复序列（LTR）控制下的细菌氯霉素乙酰转移酶（CAT）报告基因，用于比较各种载体蛋白的转染效率。组蛋白H1介导的转染产生的CAT活性为每10（6）个细胞1.66单位，是组蛋白亚组中最高的。半乳糖基化的组蛋白H1也比去唾液酸血清类黏蛋白-聚赖氨酸有效11倍。通过半乳糖基化反应，10个半乳糖基单元连接到组蛋白H1上。本报告中研究的各种蛋白质之间半乳糖基化程度的差异并不能解释不同的转染效率。用半乳糖氧化酶处理可消除半乳糖基化组蛋白H1和去唾液酸血清类黏蛋白的转染能力。组蛋白作为受体靶向DNA载体所特有的内在DNA结合结构域和核定位信号序列，有利于有效的基因递送。
